Based on sound evidence from controlled animal studies, in vivo donor pretreatment with cyclophosphamide (CY) has resulted in prolonged cadaveric renal allograft survival in several uncontrolled human studies. Significant prolongation of graft survival has been obtained in the canine model by using cryoprecipitated plasma (CPP) prepared from CY pretreated donors to perfuse renal allografts in vitro. In extension of these studies, we plan to use the active metabolite of CY, namely 4-hydroxycyclophosphamide (4-HCY) to change canine renal allograft immunogenicity in vitro by adding it to cryoprecipitated plasma for perfusion of grafts. 4-HCY is generated in aqueous solution from 4-hydroperoxycyclophosphamide (4-HPCY) which can be synthesized. In a controlled canine study, concentration of 4-HPCY and its decomposition products and time of in vitro perfusion will be analyzed in their effect on graft survival. Breakdown products of 4-HPCY, e.g., aldophosphamide and 4-HCY, will be determined and quantitated by a high performance liquid chromatographic assay (HPLC) and/or a gas chromatographic-mass spectromatic (GS-MS) assay. This study seems promising, since it is the active metabolite of CY which can change graft immunogenicity in vivo and in vitro rather than CY per se, which requires the presence of the microsomal fraction of enzymes of the liver. Furthermore, it obviates the logistical difficult problem of in vivo pretreatment.